One type of RNA editing involves the conversion of adenosine residues into inosine in dsRNA through the action of ADAR (adenosine deaminases acting on RNA). Three ADAR gene family members (ADAR1-3) have been identified in humans and rodents. When it occurs within the coding sequence, A-to-l RNA editing could result in the synthesis of proteins not directly encoded by the genome, as demonstrated with transcripts of GluR ion channels and 5-HT2c serotonin receptors. However, the most common targets for A- to-l editing are non-coding RNAs that contain inverted repeats of repetitive elements located within introns and 3' UTRs. During the past 14 years, this grant has enabled us to clone, for the first time, ADAR1, the first identified member of the ADAR gene family; this in turn led to the identification and cloning of ADAR2 and ADAR3. During the current grant support period, we created an ADAR1 null mutation in mice that causes widespread apoptosis and consequent embryonic lethal phenotypes. Our results suggest that ADAR1 is critically important for survival of numerous tissues by editing a currently unkowntarget dsRNA(s). We also demonstrated that homodimer formation of ADAR is essential for the site-selective A-to-l editing mechanism. Finally, our recent studies revealed that ADAR1 avidly binds siRNA and limits its efficacy, while both ADAR1 and ADAR2 edit specific adenosine residues of certain miRNA precursor dsRNAs, exposing the interaction of the RNAi and RNA editing pathways in mammalian cells. In this renewal application, we propose to: 1) Investigate the functional interaction of the two monomers of homodimeric ADAR for site-selective A-to-l editing and binding of siRNAs using site-directed mutagenesis of the ADAR functional domains, 2) Clone and identify target dsRNAs specifically bound by ADAR1 that may be involved in regulation of apoptosis, 3) Determine critical steps affected by A-to-l RNA editing of miRNA precursors by analyzing in vitro processing of edited miRNA precursors using Drosha and Dicer proteins or their complexes with associated factors and in vivo processing and nuclear export in transfected cells. The overall goal of this project is to elucidate the molecular mechanism of A-to-l RNA editing and to better understand its biological significance. Our research proposal will generate important information on regulation of apoptosis, which is highly relevent to many human diseases including cancer.